Human papillomaviridae comprise a large family of DNA viruses tropic for mucosal or cutaneous epithelium. Infections by these extremely prevalent pathogens can result in epithelial hyperproliferation, including ano-genital warts and laryngeal papillomas. Infections by the high-risk (HR) mucosal genotypes such as HPV-16 and HPV-18 and closely related types can progress to high grade dysplasias and cancers, notably cervical, penile, anal, and a fraction of head and neck carcinomas. Globally, over 1000 people, primarily women, die each day of HPV diseases. Prophylactic vaccines to HPV-6, -11, -16 and -18 effectively prevent new infections by these four virus types, but there are no therapeutic agents that are consistently efficacious and specific against papillomaviruses. HPVs infect basal keratinocytes, while the productive phase occurs only in the differentiated strata of squamous epithelia. We demonstrated that E7 destabilizes p130 (a member of the pRB family of pocket proteins) in the differentiated keratinocytes, enabling the S phase reentry and progression necessary for viral DNA amplification. The HR HPV E6 is known to degrade the major tumor suppressor p53 and a number of additional host proteins. The transmembrane protein E5 up-regulates the signal transduction of the ErbB family of receptor tyrosine kinases. Thus, HR HPV E7 and E6 together can immortalize primary human keratinocytes (PHKs), and E5 increases the efficiency. But how these E5 and E6 activities are utilized to promote the viral productive program remains elusive. This is because genetic analyses of HR HPVs have been conducted for well over a decade in organotypic raft cultures of immortalized epithelial cells that support viral productive program inefficiently. The limitation is compounded by the inability of E6 mutants to persist in PHKs or to immortalize them. We recently established a simple, effective, and reproducible method to generate HPV genomic plasmids in PHKs by using Cre-loxP mediated excisional recombination in vivo. A robust productive program was recapitulated by the wild type HPV-18 genome in PHK raft cultures, and progeny virions were passaged for the first time in raft cultures of naive PHKs. We validated the utility of our system for HPV genetic analyses by successfully examining an E6 mutant genome, not previously possible. Our results showed that a full-length E6 protein is necessary for efficient viral DNA amplification. Importantly, this approach circumvents the need for the immortalization functions of the HR HPVs or for pre-immortalized epithelial cell lines in order to perform genetic analyses of HPVs in PHK raft cultures. Preliminary studies revealed that E5 is also critical. We have formulated specific hypotheses on the possible roles of E5 and E6 proteins and propose to exploit this PHK raft culture system to test our hypotheses and elucidate their functions in the viral productive program. Complementary genetic and biochemical approaches will be used throughout. With a thorough understanding of the pathways which HPVs dysregulate and to which HPVs are addicted, targets of therapeutic agents will be identified to serve as the foundation for antiviral drug discovery.